Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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U-Wp-2661 - Ribi
aACKGROUND OF THE INVENTION
.
The present invention is directed to a pyridine-soluble
extract of a microorganism which, when combined with cell wall
~keleton (CWS), provides a pharmaceutical composition possessing
antl-tumor proper~ies.
; Bacteria such as Corynebacterium parvum have been the
subject of experimental work to isolate and characterize the
~omponent responsible for inducing inhibition of tumor growth
[see, fos example, Anti Tumor ActivitY and Lymphoreticular
Stimulation Properties of Fractions Isolated from C parvum;
Ca~trell, et al, Cancer Research 39~ pgs. 3554-3563 (September,
1979~]. Apart from anti-tumor activity, ~ has shown to
be a potent stimulator of the lymphoreticular system resulting in
undesirable increases in spleen and liver weigh~s and blas~o-
genesis. Applicant has discovered that a pyridine-soluble
extract of microorganism possesses potent anti-tumor properties
without the undesirable toxic effects associated with the prior
art products.
Cell wall skeleton is essentially cell wall which has had
much of the protein and lipids normally found in the cell wall
removed. It is a polymeric mycolic acid arabinogalactan
mucopeptide containing remnants of trehalose mycolate~ ("P3") and
undigested tuberculoproteinsO Cell wall skeleton is obtained
from any microorganism including, but not limited to,
M.smeqmatis, ~phlei, Nocardia rubra, Nocardia asteroides,
Corynebacterium diphtheria, Corynebacterium ~arvum, M.kansasii,
M.tuberculosis ~Strain H 37 RV and Ayoma B), and bovis Strain
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BCG. Additionally, cell wall skeleton may be obtained from such
other microorganisms as E.coli, B.abortus and Coxiella burnettii.
Cell wall skeleton may be produced by first growing and
harvesting bacteria such as M.bovis Strain BCG (Bacillus Calmette
- ~uerin). The resulting whole cell residue is processed through
a cell fractionator lRibi Cell Fractionator (Sorvall, Model
RF~ which disrupts the cells, separating the outer envelope or
cell wall from the pcotoplasmic impurities. The resulting cell
walls are then subjected to a series of solvent extractions and
enzymatic treatments (e.g., trypsin and/or chymotrypsin) to give
purified cell wall skeleton.
It is, therefore, an object of the present invention ko
provide a pharmaceutical composition containing a
pyridine-soluble extract of a microorganism in combina~ion with
cell wall skeletonO
It i~ another object of the invention to provide a method
of producing the pyridine-soluble extract of a microorganism.
It i~ still another object oE the invention to provide a
method of treating tumors in warm blooded animals and humans
using the composition containing the pyridine-soluble extract of
a microorganism and cell wall skeleton.
SUMMARY OF THE INVENTION
The present invention relates to pharmaceutical composi-
tions comprising a pyridine-soluble extract of a miccoorganism,
containing between about 7 and 20~ by weight of protein and
about 10 to 16~ by weight of sugar, and about 35 to 55% by weight
of fatty acids in combination with cell wall skeleton (CWS). The
extract preferably contains about 12% by weight of each of
protein and sugar and about 45% by weight of fatty acids.
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Any microorganism may be used to obtain the
pyridine-soluble extract including/ for example, M. bovis BCG, M.
~hlei, M. smegmatisf M. kansasii, Nocardia rubra, Corynebacterium
diphtheriae and Corynebacterium parvum. Corynebacterium ~arvum
i5 especially preferred~
Whole cells of the microorganism, preferably in the form
of a paste, ar~ mixed with pyridine. The resulting mixture is
separated to obtain a supernatant fraction which contains the
pyridine-soluble extract and a pyridine residue. Optionally, the
pyridine residue may be subjected to repeated separation proce-
dure~ as described above using pyridine to remove further
quant~ties of the desired extract.
The pyridine is then removed from the extract and the
dried extract is dialyzed agains~ a suitable liquid such as dis-
tilled water. The absence of whole cells or cell fragment
contaminants is conirmed by electron microscopy. ~he resulting
purified extract may then be lyophilized by known methods to
obtain a stable product.
The pyridine soluble extract produced in accordance with
this invention may be combined with CWS to produce a composition
having potent anti-tumor activity without stimulating the
induction of spleen and liver enlargements. The cancers which
may be ~reated by this composition include animal tumors such as
bovine squamous cell carcinoma, bovine fibrosarcoma, equine
sarcoid, eguine melanoma~ equine squamous cell carcinoma, canine
mammary tumors, canine adenoma and canine melanoma and human
tumors such as breast tumors, lung tumors, colon tumors,
malignant melanoma! squamous cell carcinomas and ovarian tumors.
The composition is preferably administered by injection in
a pharmaceutically acceptable medium such as an oil-droplet
emulsion directly into the tumor under conditions more
particularly described below. The a~oresaid composition may be
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stabilized as for example, by a lyophilization procedure and then
reconstituted without loss of potency.
The amount of the pyridine-soluble extract in a single
injection ~or the treatment of animals is between about 375 and
2500 micrograms/milliliter. The amount of CWS is between about
125 and 375 micrograms/milliliter.
The number of milliliters of the biologic injected into
the tumor is determined by the size of the tumor in accordance
with the following table:
Animal Dosage According to Tumor Size
Diameter of Tumor (cm) Amount of Biologic
Injected (ml)
0-1 up to 0.5
1-2 0.5 ~o 2.5
2-3 2.5 to 5
3-5 5 to 10
5-8 10 to 15
greater than 8 15 to 20
The maximum dose per injection is about 40 milligrams for each of
the pyridine-soluble extract and CWS. The course of treatment
comprises up to six injections administered at about two week
intervals.
The present composition in a suitable injection medium
such as an oil-droplet emulsion is administered directly into
human tumors. The amount of the pyridine~soluble extract in a
single injection is be~ween about 200 and 5000 micr~ograms, pre-
ferably between about 800 and 1200 micrograms, whiie the amount
of CWS is be~ween absut 50 and 2000 micrograms. The-preferred
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sin~le dosage level for CWS is beL~een about 475 and 525 micro-
grams. All of the above-mentioned dosage levels are based on a
typical 70 kilograms adul~ patier,t. The injections are
administered about once every week for up to a total of 15
injections.
As described above the composition for treatment of warm
blooded animals and humans may be used in the form of an oil
droplet emulsion. The amount of oil used is in the range of
between about 0.5 and 3. n percenL by volume based on the total
volume of the composition. It is preferred to use between about
0.75 and 1.5 percent by volume of the oil. Examples of such oils
include ligh~ mineral oil, squalane, 7-n-hexyloctadecane, Conoco
superoil and Drakeol 6 VR mineral oil (trademarks Eor oil,
produced by the Pennreco Company, Butler, Pennsylvania)~
The homogeni~ed oil contairling mix~ure is then combined
with a detergent which may optionally be dissolved in a saline
solution prior to mixing. The amount of detergent is typically
between about 0.02 and 0.25 percent by volume and preferably
between about 0.10 and 0.20 percent by volume based on ~he total
volume of the composition. Any common detergent material may be
used including Tween-80, and Arlacel (trademarks for products
produced by the Atlas Chemical Company).
The mixture resultir,g from the addition of detergent is
then homogenized to form a suspension which has a high percentage
o~ oil droplets coated with the active componerlts as determined by
observation under a microscope.
The following examples are for illustrative purposes
only and are not intended to limit or in any way redefine the
invention as claimed in the claims appended hereto.
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EXAMPLE 1 - Preparation of Pyridine-Soluble Extract Erom
Corynebacterium Parvum
59~Y1~1~95~8~e~59~ (P-ac~es, Strain 4182) was grown
and harvested at 37 C. in NIH thioglycolate broth for between
48 and 72 hours to obtain a whole cell paste. The paste was then
washed with 500 ml of distilled water. 90 grams ~wet weight) of
the washed paste was mixed with 200 ml. of neat pyridine and
centrifuged at 1700 x 9 for one hour at 4 C. A pyridine-soluble
extract was removed as a supernatant fraction. The remaining
residue was extracted with additional pyridine under identical
conditions as described above. Following filtration, using
Whatman No. 1 paper, the pyridine extracts were pooled and the
solvent was removed by evaporation at 50 C. in a ~uchi Rotavapor
~Brinkmann Instruments, Westbury, New York). The dried pyridine
extract was extensively dialyzed against distilled water and then
lyophilized. The resulting purified pyridine extract contained
about 12% by weight of protein, about 12~ by weight of sugar and
about 45% by weight of fatty acids. The extract was examined
under an electron microscope and found to be free of contaminat~
ing whole cells and cell wall fragments. The yield of the
pyridine-soluble extract was 9% (8.1 g.).
EXAMPLE 2 - Preparation of Pyridine-Soluble Extract from M.bovis
Strain BCG
M~ bovis strain ~CG was grown and harvested in Sautons
medium at 37Co for between 3 4 weeks to oktain a washed whole
cell paste. 50 grams (wet weight) of the washed paste was then
treated in the same manner as Example 1 to produce a yield of the
pyridine-soluble extract of 7% ~3~59)~ The extrac~ contained 15%
by weight of protein, 10% by weight of sugar and 52~ by weight of
~ZOgl5~g~
of fatty acids.
EXAMPLE 3 - Guinea-Pig Line-10 Tumor Tests
Seven strain 2 guinea pigs having Line-10 tumor growths of
about 9mm in diameter were injec~ed once with 0.4 ml of a sterile
oil droplet emulsion, i.e., Drakeol 6 VR mineral oil
tPennsylvania Refining Company, Butler, Pennsylvania), containing
300 micrograms of the pyridine-soluble extract prepared in accor-
dance with Example 1 and 50 micrograms of cell wall skeleton,
directly into the tumor tissue.
After three months, the animals were examined and in 6 of
the 7 animals, total regression had occurred.
In a control experiment, six strain 2 guinea pigs having
Line-10 tumor growths of about 9mm in diameter were injected once
with 0.4 ml of the sterile oil droplet emulsion described above
without the pyridine extract or cell wall skeleton. The
injections were made directly into the tumor tissue. None of the
six tumors showed any signs of regression after three months.
